Core binding factors (CBFs) are heterodimeric transcription factors consisting of DNA-binding RUNX (AML1) subunits and a non-DNA-binding CBFB subunit. All four genes that encode RUNX subunits (RUNX1, RUNX2, RUNX3) and the gene encoding the CBFB subunit are essential for normal development, and are mutated in human disease. RUNX1 and CBFB are required for hematopoiesis, and are mutated in 25%-30% of human leukemias. RUNX1 and CBFB are proto-oncogenes commonly activated in human leukemias. The reversion and translocations identified in these genes are associated with ~30% of de novo acute myeloid leukemias (AML) in humans. The t(8;21) associated with 12-15% of AML cases generates a novel fusion protein, AML1-ETO, containing the N-terminal 177 amino acids of RUNX1 including the Runt domain and virtually all (577 amino acids) of ETO. Heterozygous knock-in of AML1-ETO was lethal indicating that it is a dominant negative. An inducible mouse model of AML1-ETO shows that it highly predisposes mice to the development of leukemia. We are proposing to test the role of 2 functional domains of AML1-ETO. Aim 1: Structural characterization of functional domains of AML1-ETO (HHR and MYND domains) to test the hypothesis that each domain is essential for the dominant negative phenotype of AML1-ETO. Aim 1 proposes to solve the structures of these two domains (HHR and MYND) by themselves or as functional complexes using NMR spectroscopy or x-ray crystallography. Aim 2: Biochemical characterization of functional domains of AML1-ETO (HHR and MYND domains) to test the hypothesis that each domain is essential for the dominant negative phenotype of AML1-ETO. Aim 2 proposes to measure the binding constants of these individual domains and longer forms with interacting proteins and with DNA using isothermal titration calorimetry and surface plasmon resonance methods, including identification of hot spots for interaction by Ala point mutagenesis. Aim 3: In vivo characterization of functional domains of AML1-ETO (HHR and MYND domains) to test the hypothesis that each domain is essential for the dominant negative phenotype of AML1-ETO. Based on the identification of highly specific point mutations that disrupt particular interactions of AML1-ETO, we can surgically test the role of each of these functions in vivo.